Chronic accumulation of myofibroblasts in healing wounds is associated with hypercontractility, excessive deposition of interstitial collagens, and destructive tissue remodeling. Vascular smooth muscle alpha-actin ((-SMA) is a contractile protein transiently expressed by differentiated myofibroblasts for generating tensile force required to close open wounds. In chronic fibrotic disease, myofibroblast differentiation is dysfunctional and we discovered that molecular signaling required for activation of both the (-SMA and type I collagen genes in these cells also provides negative feedback that could potentially limit the recruitment of hyperactive myofibroblasts during wound healing and destructive remodeling. Studies outlined in this proposal are expected to reveal novel forms of functional interplay of the DNA- and mRNA-binding proteins YB-1, Pur (, and Pur ( with the (-SMA and type I collagen promoters and clarify how these proteins are affected by pro- fibrotic agents such as TGF(1 and thrombin that, if unchecked, may cause myofibroblast progression to hypertrophic scarring. Experiments are designed to initiate, amplify, or attenuate myofibroblast differentiation to better understand strategies for controlling (-SMA and type I collagen gene output at the transcriptional and translation levels as well as reveal novel interventional strategies that might be useful for minimizing aberrant wound healing outcomes. Aim 1 will examine TGF(1-regulated interaction of YB-1 and Pur protein repressors with (-SMA and collagen promoter DNA and the transcriptional activators Sp1, SRF, and Smads 2,3, delineate regions of repressor polypeptide chains required for this functional interplay, and attempt to disrupt complex formation and disable pathobiologic myofibroblast differentiation using peptide decoys and small molecule pharmacologic inhibitors. Aim 2 will determine if thrombin potentiates myofibroblast differentiation at the level of translational control thus functioning as a TGF(1 supplement or instead antagonizes this growth factor by blocking transcription and myofibroblast recruitment by inducing the anti-fibrotic transcriptional regulatory protein, Egr-1. Aim 3 studies will explore alternative, Smad-independent mechanisms of myofibroblast differentiation and fibrosis. Loss-of-function approaches based on pharmacologic inhibition of TGF(1/Smad kinase- or phosphatidylinositol-3-kinase (PI3K)/Akt kinase signaling will be used to evaluate their possible suppressive effect on myofibroblast activation in vitro and cardiac fibrosis in mice after ischemia/reperfusion injury. The ability of TGF(1 and thrombin to exploit the unique DNA-, RNA-, and protein-binding properties of YB-1 and Pur proteins adds a new dynamic perspective to control of gene expression during myofibroblast differentiation that may reveal optimum strategies for therapeutic management of chronic fibrotic diseases. Project Narrative: The proposed research investigates cellular and molecular changes in connective tissue cells in heart and lung tissues that are required for normal wound healing after surgery. In transplant patients as well as individuals diagnosed with various forms of chronic cardiopulmonary diseases, these processes are accelerated leading to permanent scar formation, structural deformities in the heart and lung, loss of tissue function, and early death. New research studies outlined in this application will examine the individual biochemical pathways that drive normal wound healing and attempt to identify new targets for drug therapy that will help control destructive wound healing activity and improve the quality of life for individuals suffering from chronic diseases that cause abnormal scar tissue formation in the heart and lung. [unreadable] [unreadable] [unreadable]